Antigenic differences in the surface mannoproteins of Candida albicans as revealed by monoclonal antibodies.

Monoclonal antibodies to Candida albicans were prepared with blastoconidia bearing germ tubes used as the immunogen. Four antibodies reacted by immunofluorescence with surfaces of C. albicans as well as Candida stellatoidea, Candida tropicalis, and several strains of C. albicans, but not with Torulopsis glabrata. One antibody reacted with Saccharomyces cerevisiae. In addition, the monoclonal antibodies precipitated material of approximately 200 kilodaltons when tested against metabolically labeled blastoconidia digests. The monoclonal antibodies exhibited heterogeneous staining of C. albicans surfaces, as shown by immunofluorescence. None of the monoclonal antibodies were specific to germ tubes. More importantly, however, two of the monoclonal antibodies reacted with the mannoprotein precipitin arc of C. albicans that was produced by reference rabbit polyclonal antisera by crossed immunoelectrophoresis, thus linking the heterogeneity seen by immunofluorescence to the heterogeneity in mannoproteins. Finally, three of the monoclonal antibodies reacted with a glycan fraction of cell digests, indicating their reactivity with the carbohydrate portion of the mannoprotein.

Transformation by the oncogene v-fms: the effects of castanospermine on transformation-related parameters.

The effects of castanospermine on various parameters associated with transformation were examined in cells expressing the viral oncogene v-fms. Fischer rat embryo (FRE) cells transformed by the oncogene v-fms and grown in the presence of castanospermine reverted to a more normal cell morphology and accumulated fms protein within the endoplasmic reticulum. Treated cells attained contact inhibition of cell growth at a much lower cell density compared to the untreated controls. No effect of castanospermine on cell growth was observed for FRE cells transformed by a different oncogene v-fgr. Castanospermine-treated SM-FRE (v-fms transformed) cells reexpressed extracellular matrix fibronectin and exhibited an extensive actin-containing cytoskeleton similar to that of normal nontransformed FRE cells. Castanospermine treatment of SM-FRE cells resulted in a sixfold decrease in [3H]deoxyglucose uptake compared to that of the nonreverted SM-FRE cells. Again, no effect was observed in FRE cells transformed by the oncogene v-fgr (GR-FRE). These results further characterize the reversion caused by castanospermine and indicate that cell surface expression coordinately controls anchorage independent growth, cell morphology, contact inhibition of growth, and hexose uptake.

Antigenic differences between mannoproteins of germ tubes and blastospores of Candida albicans.

To determine the nature of germ tube-specific antigens of Candida albicans, procedures for intrinsically labeling cell wall antigens metabolically were developed. Blastospores or germ tubes labeled either in their proteins with L-[35S]methionine or in mannose-containing carbohydrates with D[2-3H]mannose contained surface components similar to those found previously with 125I-labeled organisms. Germ tube-specific determinants, were found on a 200-kilodalton protein in digests from germ tubes, whereas a component of similar molecular size in blastospore extracts reacted weakly or not at all with germ tube-specific antibody. In addition, a glycan fraction prepared from germ tubes reacted with the unadsorbed anti-C. albicans polyvalent antibody but not with the germ tube-specific antibody, suggesting that the germ tube-specific determinants are not carbohydrates.

Domain-specific distribution of carbohydrates in human fibronectins and the transformation-dependent translocation of branched type 2 chain defined by monoclonal antibody C6.

Fibronectins purified from human plasma (termed pFN), spent culture media of human fibroblasts WI38 (termed cFN), and SV40 virus-transformed WI38/VA13 cells (termed tFN) and their cleavage fragments were compared with respect to their binding activities to lectins and anti-carbohydrate antibodies reacting with chemically well-defined structures. The following findings were of particular interest. About 25-35% of cFN and tFN carried a binary sialosyl type 2 chain (NeuAc alpha 2----3/or 6Gal beta 1----4GlcNAc) linked beta 1----3/beta 1----6 to the galactose residue and defined by monoclonal antibody C6. This structure was not detected in pFN. In cFN, the C6-defined structure was localized within the gelatin-binding domain, whereas in tFN the same structure was absent from this domain but was located at the NH2-terminal region of the central domain. Other carbohydrate determinants, defined by Ricinus communis lectin and concanavalin A before and after sialidase treatment, showed essentially identical domain distribution patterns among cFN, tFN, and pFN and were all located at the gelatin-binding domain (44 kDa), its precursor (60 kDa), and the Cell/Hep-2 domain (155/145 kDa). Although both cFN and tFN were reactive with lentil lectin, pFN was not. Fibronectin from transformed cells (tFN) showed much greater reactivity than cFN and pFN with wheat germ lectin before sialidase treatment and showed enhanced reactivity with R. communis lectin and peanut lectin after sialidase treatment, indicating that tFN is more highly sialylated than cFN and pFN. All fibronectins examined were strongly reactive with monoclonal antibody AH8-28, which binds to Gal beta 1----3GalNAc residues, and this reactivity was localized to both the NH2-terminal half and COOH-terminal half of the S-cyanylation-cleaved fibronectin molecule.

A monoclonal antibody defining a binary N-acetyllactosaminyl structure in lactoisooctaosylceramide (IV6Gal beta 1----4GlcNAcnLc6): a useful probe for determining differential glycosylation patterns between normal and transformed human fibroblasts.

Monoclonal antibodies A5 and C6 have been reported previously to recognize developmentally regulated determinants involving N-acetyllactosamine [Fenderson B. A., O'Brien D. A., Millette C. F. and Eddy E. M. (1984) Devl Biol. 103, 117-128]. In the present study, the specificity of these antibodies was determined by solid-phase radioimmunoassay and by thin-layer chromatography immunostaining using purified glycolipid standards. Antibody A5 recognized N-acetyllactosamine (type 2 chain; Gal beta 1----4GlcNAc beta 1----3R), irrespective of branching status. In contrast antibody C6 recognized the binary N-acetyllactosamine structure carried on lactoisooctaosylceramide. Antibody C6 did not react with sialosyl or alpha-galactosyl derivatives of the isooctaosyl structure, including human G10, G8 and bovine G9. Thus, unlike other anti-I antibodies, C6 provides a specific probe for both branching status and absence of terminal chain modification. Monoclonal antibodies A5, C6 and anti-I(Ma) were used to investigate glycosylation changes associated with oncogenic transformation. In contrast to results with lectins, these antibodies preferentially labeled the major glycoproteins of SV40-transformed human embryonic lung fibroblasts, including GP80, GP180, GP200 and GP250. The results suggest that increased expression of unsubstituted polylactosamine core structure at the cell surface follows SV40-transformation.

Transformation by the v-fms oncogene product: role of glycosylational processing and cell surface expression.

The effect of glycosylational-processing inhibitors on the synthesis, cell surface expression, endocytosis, and transforming function of the v-fms oncogene protein (gp140fms) was examined in McDonough feline sarcoma virus-transformed Fischer rat embryo (SM-FRE) cells. Swainsonine (SW), a mannosidase II inhibitor, blocked complete processing, but an abnormal v-fms protein containing hybrid carbohydrate structures was expressed on the cell surface. SW-treated SM-FRE cells retained the transformed phenotype. In contrast, two glucosidase I inhibitors (castanospermine [CA] and N-methyl-1-deoxynojirimycin [MdN]) blocked carbohydrate remodeling at an early stage within the endoplasmic reticulum and prevented cell surface expression of v-fms proteins. CA-treated SM-FRE cells reverted to the normal phenotype. Neither SW, CA, nor MdN affected either endocytosis or the tyrosine kinase activity associated with the v-fms gene product in vitro. These results demonstrate the necessity of carbohydrate processing for cell surface expression of the v-fms gene product and illustrate the unique ability to modulate the transformed state of SM-FRE cells with the glycosylational-processing inhibitors CA and MdN.

Carbohydrate determinants associated with carcinoembryonic antigen (CEA).

The reactivities of eight purified preparations of carcinoembryonic antigen with monoclonal antibodies directed to tumor-associated carbohydrate determinants have been studied. All eight preparations showed strong reactivities with AH6, which defines Y structure (Fuc alpha 1----2Gal beta 1----4[Fuc alpha 1----3] GlcNAc beta 1----R), whereas only a few preparations showed reactivity with FH4-defining dimeric X determinants, (Gal beta 1----4 [Fuc alpha 1----3]GlcNAc beta 1----3Gal beta 1----4 [Fuc alpha 1----3]GlcNA beta 1----3Gal beta 1----R). No other antibodies tested showed any reactivity with these preparations. These carbohydrate markers associated with carcinoembryonic antigen will be useful to enhance the diagnostic value of the antigen.

Cell surface expression of the McDonough strain of feline sarcoma virus fms gene product (gp 140fms).

The unique oncogene carried by the McDonough strain of feline sarcoma virus (SM-FeSV), called v-fms, directs the synthesis of a set of related glycoproteins, called gP 180gag-fms, gp 140fms, and gp 120fms. We have prepared antibodies to these proteins and used indirect immunofluorescence techniques on viable SM-FeSV transformed cells to demonstrate that fms-specific determinants are expressed on the external surface. The fms-specific fluorescence co-localized with clathrin and was detectable in clathrin-coated pits and endocytotic vesicles. Two cell surface labeling methods indicated that gp140fms was the only fms-related protein on the cell surface. In view of the relationship between the erbB oncogene product and the epidermal growth factor receptor, and the fact that growth factor receptors utilize clathrin-coated pits in endocytosis, we believe the gp140fms transforming protein of SM-FeSV also could function as an analog of a growth factor receptor.

Immunochemical characterization of a heat-stable surface antigen of Mycoplasma pulmonis expressing both species-specific and strain-specific determinants.

We sought to characterize the strain-specific antigens of four strains of Mycoplasma pulmonis (47, 63, Negroni, and 19612) by crossed immunoelectrophoresis. Although the strains possessed a number of common antigens, type-specific antigens of 0.32 mobility (bovine albumin was assigned a value of 1) were detected in strains 47 and 63. Strains 19612 and Negroni cross-reacted and represented a third group. Each strain possessed a major heat-stable antigen complex of 0.32 mobility characterized by a faster-moving component of 0.55 mobility. Monospecific antiserum to heat-stable antigen 0.32 of strain 63 demonstrated that this antigen complex consisted of at least three antigen-antibody precipitating systems characterized by type-specific and group-specific determinants. Adsorption of antiserum with whole cells revealed that the 0.32 antigen complex was surface exposed. The antigen complex is pronase sensitive and only partially sensitive to periodate. Purification of antigen 0.32 from detergent-extracted cells by affinity chromatography using monospecific antiserum revealed two major polypeptides of 86,500 and 83,500 dalton which reacted strongly with monospecific antiserum by immunoblotting. These reactive polypeptides were present in all strains examined. Additional polypeptides of different molecular weights in strains 19612 and Negroni produced strong reactions with monospecific antiserum, although sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles of the two strains were strikingly similar. Common heat-stable antigens were observed also. This study demonstrates that M. pulmonis strains possess an antigenically variable heat-stable surface antigen which is unique in that it contains not only strain-specific determinants but also species-specific determinants.

Glycosidic Enzyme Activity in Pea Tissue and Pea-Fusarium solani Interactions.

Membrane barriers which prevent direct contact between Fusarium solani and pea endocarp tissue prevent fungal spores from inducing phytoalexin production. Conversely, preinduced host resistance responses are not readily transported from the plant across the membrane barrier to Fusarium macroconidia.Crude enzyme extracts from pea endocarp tissues partially degrade Fusarium solani f. sp. phaseoli cell walls. Activities of the glycosidic enzymes, chitinase, beta-1,3-glucanase, chitosanase, beta-D-N-acetylglucosaminidase, beta-D-N-acetylgalactosaminidase, beta-D-glucosidase, alpha-D-glucosidase, and alpha-D-mannosidase, were detected in pea endocarp tissue. If pods are challenged with Fusarium spores or chitosan, the chitinase activity of the infected tissue remains higher than water-treated pods 0.5 to 6 hours after treatment. The beta-1,3-glucanase activity increases within 6 hours in both inoculated and control tissue. Chitosanase activity was lower in tissue treated with Fusarium solani f. sp. pisi, f. sp. phaseoli or chitosan than in water-treated control tissue. Thus, the pea tissue contains glycosidic enzymes with the potential to degrade the major compounds of the Fusarium cell walls.